Submersed macrophytes have important ecological roles but non-native invasive species may affect biodiversity and water uses. We investigated the native macrophyte Egeria najas and the invasive Hydrilla verticillata and measured their maximum colonization depth and its relationship with Secchi disk depth, their biomass along the depth gradient and their preferred depths of occurrence. The Itaipu Reservoir was monitored for seven years, during which maximum colonization depth and Secchi disk depth were measured. During a separate sampling, plants were collected to determine biomass along the depth gradient. Ancova showed that the maximum colonization depth of both species increased with increasing Secchi disk depth, but the maximum colonization depth of H. verticillata increased faster with increasing water transparency than did that of E. najas. Quadratic regression revealed that the biomass of each species peaks at intermediate depths. Hydrilla verticillata colonizes deeper regions than does E. najas. The patterns found in the present study can be explained by underwater light and, probably, wave disturbances. The preference of H. verticillata for deeper sites indicates that the ecological niches of the two macrophytes differ, and that H. verticillata has great potential to spread and accumulate biomass in reservoirs.